Description of the project. Viruses are the ultimate intracellular parasites. With minimal genetic resources they are able to reroute cellular metabolic pathways and hijack cellular proteins to promote their own replication. Thus, understanding the role of specific host factors in the viral life cycle is essential for the development of novel anti-viral strategies. Enteroviruses are a group of picornaviruses, small +RNA viruses of vertebrates including humans. Enteroviruses cause clinically and economically important human diseases, ranging from the common cold to fatal encephalitis and myocarditis. High genetic diversity and adaptability of these viruses complicates development of comprehensive vaccines and traditional therapeutics targeting virus-specific proteins. Of all the pathogenic enteroviruses, only polioviruses can be controlled with vaccines, and no clinically approved anti-enterovirus drugs are available. Thus, novel approaches are urgently needed to control enteroviruses associated with human diseases. Picornaviruses replicate their genomes on specialized membrane domains, replication organelles that feature unique lipid and protein composition and whose development relies on the re- organization of cellular lipid synthesis and membrane trafficking pathways. This implies that at least some cellular membrane metabolism components must be indispensable for viral propagation. Indeed, enteroviruses universally require the host protein GBF1 for their replication. GBF1 is a large multi-domain protein that functions as a guanine nucleotide exchange factor (GEF) for select small GTPases of the Arf family. GBF1 is a master coordinator of the early steps of protein transport in the secretory pathway, and participates in maintaining Golgi structure and function and in lipid droplet metabolism. However, how GBF1 supports the viral replication cycle remains unknown. Attempts to relate the known cellular activities of GBF1 such as Arf activation, membrane remodeling and recruitment of cellular proteins to membranes to GBF1 function in viral replication produced controversial results. GBF1 is known to interact with numerous cellular proteins and with the enterovirus replication protein 3A, but how viruses use these interactions to their advantage remains unknown. Herein, we propose a new model in which GBF1 acts as a molecular scaffold to coordinate the assembly of viral and cellular proteins into operational replication complexes. For this project, we built a team of a cell biologist with a superior expertise in GBF1 biology (Sztul), and a virologist with an outstanding background in picornavirus replication (Belov). Together, we will delineate the step(s) in viral life cycle that require GBF1 and uncover the mechanisms of GBF1 action in the formation function of replication complexes. The universal reliance of diverse enteroviruses on GBF1 provides an unprecedented opportunity for the development of broad-spectrum therapeutics targeting GBF1-controlled processes in infected cells.